The long-term objective of the proposed project is to elucidate the role of mechanotransduction in bone adaptation. Depending on their substrate geometries, osteoblasts grow and develop differently in the in vitro culturing system. Compared to the cells cultured on 2D surface, osteoblasts grown in a 3D matrix exhibit slower cellular proliferation and enhanced calcium deposition. The observed difference raises a question on the role of substrate geometries in the osteoblastic responses to mechanical stimuli. Our current in vitro knowledge on mechanotransduction is mostly based on the osteoblasts grown on a flat substrate. We recently developed a novel mechanical loader with a piezoelectric actuator suitable for applying strain and fluid flow to osteoblasts grown in the 3D porous collagen matrix. The mechanical loader is capable of generating strain in any waveform and strain-induced fluid flow in tubular collagen pores in the matrix. Using this mechanical loader, the proposed project aims to investigate the responses of osteoblasts in the 3D collagen matrix, and the role of intercellular contacts in mechanotransduction, especially the role of gap junctions and actin filaments. Three specific aims of the proposed project are to (i) identify the specific waveforms most effective in inducing mechanotransduction (ii) compare the responses to strain, strain-induced fluid flow, and global chemotransport of osteoblasts grown in the 2D and 3D culturing systems, and (iii) examine the role of gap junctions and actin filaments in mechanotransduction in the 3D matrix. In assaying the responses to mechanical stimuli, mRNA levels, protein levels, and proteolytic activities of the genes sensitive to strain/ stress will be determined using the strain waveform measured in dogs during walking. The mRNA level will be determined by cDNA array as well as RT-PCR, and the protein level will be determined by Western analysis. Activity of matrix metalloproteinases, a family of proteolytic enzymes responsive to mechanical stimuli, will be assayed using fluorescent fibril degradation and zymography. Pharmacological inhibitors and dominant negative mutants will be used to disrupt intercellular communications. Completion of these studies should provide significant insight into the mechanical responses of osteoblasts in a 3D environment, and contribute to developing. q load-induced in vitro connective tissue engineering.